Lactic acid bacteria relate to a group of Gram-positive, non-sporing bacteria, which carry out a lactic acid fermentation of sugars.
Lactic acid bacteria are commercially widely used for instance in the preparation of different food products such as e.g. yogurts.
Further, many lactic acid bacteria are probiotics—i.e. live microorganisms which when administered in adequate amounts confer a health benefit on the host (e.g. a human).
A lactic acid bacteria product is commercially often sold as a dried composition—e.g. a freeze dried composition. The dried composition may e.g. be dried pellets or a tablet (e.g. made from milled dried pellets).
Lactic acid bacteria (e.g. dried) to be used for human or animal consumption are frequently formulated with ascorbic acid or ascorbate such as e.g. sodium ascorbate as antioxidant—sodium ascorbate e.g. improves the storage stability of the lactic acid bacteria product.
Sodium ascorbate is a salt of ascorbic acid (vitamin C) and it has the common E-number E301 (see EU food additive legislation). The structures of sodium ascorbate and ascorbic acid are shown in FIG. 1 herein.
The article—L. Kurtmann et al., “Storage stability of freeze-dried Lactobacillus acidophilus (La-5) in relation to water activity and presence of oxygen and ascorbate”; Cryobiology (2009), doi:10.1016/j.cryobio1.2008.12.001—was published on the Internet in December 2008.
The L. Kurtmann et al article describes that when sodium ascorbate was present a pink/red color was observed on the surface of dried Lactobacillus acidophilus compositions/samples during storage (see e.g. the abstract).
Such a pink/red color is unwanted—for instance many consumers do not like such a pink/red color since it may give an un-healthy “look” of the dried lactic acid bacteria product.
During fermentation lactic acid bacteria makes lactic acid—accordingly in order for not getting a too low pH during production the pH is controlled during fermentation by addition of a base.
Generally, the pH is controlled in order to have a 3≦pH≦8 during at least the majority of the fermentation process, since lactic acid bacteria generally do not grow properly at pH below 3.
In the L. Kurtmann et al article L. acidophilus (La-5®) from Chr. Hansen A/S was used. In the article it is generically stated that pH was controlled during fermentation (see Material and methods). No mention is made about how the pH control was carried out.
Use of NH3 as base is very normal for commercial industrial relevant production of lactic acid bacteria products in industry—in fact it may be considered as a “standard”.
Page 179, right column of the L. Kurtmann article discussed above indicates that amino groups from the bacterial cells or from fermentation residues could be involved in the creation of the unwanted pink/red color—the article reads “a red compound is formed when ascorbic acid is oxidized to dehydroascorbic acid and reacts with amino groups forming the pigment”.
As further discussed below—in fact one may say that this disclosed theory of the L. Kurtmann article TEACHES AWAY from the present invention (i.e. NOT using a base comprising NH3).
EP-A-1038951 (Nestle [CH]) discloses a media for cultivating bacteria, the media comprising ascorbic acid as an antioxidant and buffers like Na2CO3, KH2PO4 (see page 3, claims 3 and 4).
In [0026] it is said that e.g. ascorbic acid is provided simply as an example of a suitable antioxidant comprises in the fermentation MEDIUM.
Said in other words, EP-A-1038951 do not directly and unambiguously disclose a method for making bacteria, wherein ascorbic acid is added to the harvested bacteria concentrate—i.e. as discussed below, EP-A-1038951 does not explicitly disclose step (iii) of first aspect (claim 1) of the present invention as described herein.